A Small Compound, HYGIC, Promotes Hypocotyl Growth Through Ectopic Ethylene Response.

Plant seedlings adjust the growth of the hypocotyl in response to surrounding environmental changes. Genetic studies have revealed key players and pathways in hypocotyl growth, such as phytohormones and light signaling. However, because of genetic redundancy in the genome, it is expected that not-yet-revealed mechanisms can be elucidated through approaches different from genetic ones. Here, we identified a small compound, HYGIC (HG), that simultaneously induces hypocotyl elongation and thickening, accompanied by increased nuclear size and enlargement of cortex cells. HG-induced hypocotyl growth required the ethylene signaling pathway activated by endogenous ethylene, involving CONSTITUTIVE PHOTOMORPHOGENIC 1, ETHYLENE INSENSITIVE 2 (EIN2) and redundant transcription factors for ethylene responses, ETHYLENE INSENSITIVE 3 (EIN3) and EIN3 LIKE 1. By using EBS:GUS, a transcriptional reporter of ethylene responses based on an EIN3-binding-cis-element, we found that HG treatment ectopically activates ethylene responses at the epidermis and cortex of the hypocotyl. RNA-seq and subsequent gene ontology analysis revealed that a significant number of HG-induced genes are related to responses to hypoxia. Indeed, submergence, a representative environment where the hypoxia response is induced in nature, promoted ethylene-signaling-dependent hypocotyl elongation and thickening accompanied by ethylene responses at the epidermis and cortex, which resembled the HG treatment. Collectively, the identification and analysis of HG revealed that ectopic responsiveness to ethylene promotes hypocotyl growth, and this mechanism is activated under submergence.

Tip60/KAT5 Histone Acetyltransferase Is Required for Maintenance and Neurogenesis of Embryonic Neural Stem Cells.

Epigenetic regulation via epigenetic factors in collaboration with tissue-specific transcription factors is curtail for establishing functional organ systems during development. Brain development is tightly regulated by epigenetic factors, which are coordinately activated or inactivated during processes, and their dysregulation is linked to brain abnormalities and intellectual disability. However, the precise mechanism of epigenetic regulation in brain development and neurogenesis remains largely unknown. Here, we show that Tip60/KAT5 deletion in neural stem/progenitor cells (NSCs) in mice results in multiple abnormalities of brain development. Tip60-deficient embryonic brain led to microcephaly, and proliferating cells in the developing brain were reduced by Tip60 deficiency. In addition, neural differentiation and neuronal migration were severely affected in Tip60-deficient brains. Following neurogenesis in developing brains, gliogenesis started from the earlier stage of development in Tip60-deficient brains, indicating that Tip60 is involved in switching from neurogenesis to gliogenesis during brain development. It was also confirmed in vitro that poor neurosphere formation, proliferation defects, neural differentiation defects, and accelerated astrocytic differentiation in mutant NSCs are derived from Tip60-deficient embryonic brains. This study uncovers the critical role of Tip60 in brain development and NSC maintenance and function in vivo and in vitro.

Affinity-based high-resolution analysis of DNA binding by VASCULAR-RELATED NAC-DOMAIN7 via fluorescence correlation spectroscopy.

VASCULAR-RELATED NAC-DOMAIN7 (VND7) is the master transcription factor for vessel element differentiation in Arabidopsis thaliana. To identify the cis-acting sequence(s) bound by VND7, we employed fluorescence correlation spectroscopy (FCS) to find VND7-DNA interactions quantitatively. This identified an 18-bp sequence from the promoter of XYLEM CYSTEINE PEPTIDASE1 (XCP1), a direct target of VND7. A quantitative assay for binding affinity between VND7 and the 18-bp sequence revealed the core nucleotides contributing to specific binding between VND7 and the 18-bp sequence. Moreover, by combining the systematic evolution of ligands by exponential enrichment (SELEX) technique with known consensus sequences, we defined a motif termed the Ideal Core Structure for binding by VND7 (ICSV). We also used FCS to search for VND7 binding sequences in the promoter regions of other direct targets. Taking these data together, we proposed that VND7 preferentially binds to the ICSV sequence. Additionally, we found that substitutions among the core nucleotides affected transcriptional regulation by VND7 in vivo, indicating that the core nucleotides contribute to vessel-element-specific gene expression. Furthermore, our results demonstrate that FCS is a powerful tool for unveiling the DNA-binding properties of transcription factors.

Visualization and isolation of zone-specific murine hepatocytes that maintain distinct cytochrome P450 oxidase expression in primary culture.

Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/ß-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zone-specific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development.

Transcription Factors VND1-VND3 Contribute to Cotyledon Xylem Vessel Formation.

Arabidopsis (Arabidopsis thaliana) VASCULAR-RELATED NAC-DOMAIN1 (VND1) to VND7 encode a group of NAC domain transcription factors that function as master regulators of xylem vessel element differentiation. These transcription factors activate the transcription of genes required for secondary cell wall formation and programmed cell death, key events in xylem vessel element differentiation. Because constitutive overexpression of VND6 and VND7 induces ectopic xylem vessel element differentiation, functional studies of VND proteins have largely focused on these two proteins. Here, we report the roles of VND1, VND2, and VND3 in xylem vessel formation in cotyledons. Using our newly established in vitro system in which excised Arabidopsis cotyledons are stimulated to undergo xylem cell differentiation by cytokinin, auxin, and brassinosteroid treatment, we found that ectopic xylem vessel element differentiation required VND1, VND2, and VND3 but not VND6 or VND7. The importance of VND1, VND2, and VND3 also was indicated in vivo; in the vnd1 vnd2 vnd3 seedlings, xylem vessel element differentiation of secondary veins in cotyledons was inhibited under dark conditions. Furthermore, the light responsiveness of VND gene expression was disturbed in the vnd1 vnd2 vnd3 mutant, and vnd1 vnd2 vnd3 failed to recover lateral root development in response to the change of light conditions. These findings suggest that VND1 to VND3 have specific molecular functions, possibly linking light conditions to xylem vessel formation, during seedling development.

Increased Incidence of Visual Field Abnormalities as Determined by Frequency Doubling Technology Perimetry in High Computer Users Among Japanese Workers: A Retrospective Cohort Study.

BACKGROUND: This retrospective cohort study primarily aimed to investigate the possible association of computer use with visual field abnormalities (VFA) among Japanese workers. METHODS: The study included 2,377 workers (mean age 45.7 [standard deviation, 8.3] years; 2,229 men and 148 women) who initially exhibited no VFA during frequency doubling technology perimetry (FDT) testing. Subjects then underwent annual follow-up FDT testing for 7 years, and VFA were determined using a FDT-test protocol (FDT-VFA). Subjects with FDT-VFA were examined by ophthalmologists. Baseline data about the mean duration of computer use during a 5-year period and refractive errors were obtained via self-administered questionnaire and evaluations for refractive errors (use of eyeglasses or contact lenses), respectively. RESULTS: A Cox proportional hazard analysis demonstrated that heavy computer users (>8 hr/day) had a significantly increased risk of FDT-VFA (hazard ratio [HR] 2.85; 95% confidence interval [CI], 1.26-6.48) relative to light users (<4 hr/day), and this association was strengthened among subjects with refractive errors (HR 4.48; 95% CI, 1.87-10.74). The computer usage history also significantly correlated with FDT-VFA among subject with refractive errors (P < 0.05), and 73.1% of subjects with FDT-VFA and refractive errors were diagnosed with glaucoma or ocular hypertension. CONCLUSIONS: The incidence of FDT-VFA appears to be increased among Japanese workers who are heavy computer users, particularly if they have refractive errors. Further investigations of epidemiology and causality are warranted.

Effect of acetamiprid on the immature murine testes.

Neonicotinoids, such as acetamiprid (ACE), a pesticide used worldwide, are believed to be safe for human use. These molecules are structurally similar to nicotine, act as nicotinic acetylcholine receptor (nAChR) agonists, and were shown to be associated with neuromuscular and reproductive disorders, but these experiments were primarily performed in mature animals. In this study, the effects of ACE on the testes of immature mice were examined. The exposure of 3-week-old mice to ACE-containing water for 180 days led to a decrease in body weight and mildly affected spermatogenesis. Additionally, the expression of testosterone-metabolism genes, nAChR subunit genes, and proliferation-associated genes decreased in the testes of ACE-treated mice. Our results show that immature rodents may be less sensitive to ACE than mature ones, that mice may be more likely to accumulate ACE than rats, and that the development of disorders may be affected by the accumulation of ACE in the testes.

Developmentally Regulated RNA-binding Protein 1 (Drb1)/RNA-binding Motif Protein 45 (RBM45), a Nuclear-Cytoplasmic Trafficking Protein, Forms TAR DNA-binding Protein 43 (TDP-43)-mediated Cytoplasmic Aggregates.

Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.

The intrauterine environment affects learning ability of Tokai high avoider rat offspring derived using cryopreservation and embryo transfer-mediated reproduction.

Embryo transfer (ET) to recipient female animals is a useful technique in biological and experimental animal studies. While cryopreservation of two-cell stage rat embryos and ET to recipient rats are currently well-defined, it is unknown whether these artificial reproductive techniques and maternal factors affect offspring phenotype, particularly higher brain functions. Therefore, we assessed the effects of cryopreservation, ET, and maternal care on learning behaviour of the offspring, using Tokai high avoider (THA) rats that have a high learning ability phenotype. We found that the high learning ability of THA rat offspring was not replicated following ET to surrogate Wistar rats with a low-avoidance phenotype. Additionally, the characteristic phenotype of offspring obtained through mating of ET-derived rats was similar to that of THA rats. A postnatal cross-fostering investigation with the offspring of Wistar and THA rats showed that maternal behaviour, including postnatal care and lactation traits, did not differ between the dams of low-avoidance Wistar rats and THA rats; therefore, learning behaviour was retained in both Wistar and THA rat offspring. We conclude that the offspring phenotype, although unchanged, has an imperceptible effect on the learning ability of ET-derived THA rats through the intrauterine environment of the recipient.

[Operation for Intracardiac Angiomyolipoma;Report of a Case].

Primary tumors of the heart are rare. In the literature operations for heart tumors account for 0.3% of all cardiac surgical procedures. We report a very rare case of primary cardiac angiomyolipoma. A 39-years-old man was admitted with chest discomfort. A mass was detected in the left ventricular myocardium by enhanced chest computed tomography. We performed resection of the intramyocardial tumor under cardiopulmonary bypass. Pathologic examination revealed a 4×3×3 cm, dome-shaped, solid tumor composed of a mixture of blood vessels, smooth muscle, and fat tissue. Histopathological findings showed that the tumor was angiomyolipoma. Angiomyolipoma is a benign tumor and usually develop in the kidney and are associated with tuberous sclerosis. In our case, there was no evidence of tuberous sclerosis.

Interaction of human mitochondrial transcription factor A in mitochondria: its involvement in the dynamics of mitochondrial DNA nucleoids.

Mitochondrial transcription factor A (TFAM) is a key regulator of mitochondrial DNA (mtDNA). TFAM interacts with itself and forms dimers; however, the precise interaction domain in vivo has not yet been determined. We herein showed that human TFAM formed oligomers in mitochondria by in situ chemical cross-linking. We used the separated fluorescent protein, monomeric Kusabira-Green, as a reporter to monitor their self-association in mitochondria. This reporter successfully detected the TFAM-TFAM interaction in cells as fluorescent signals on mitochondria. We also found that the N-terminal high-mobility group box domain was sufficient for this interaction. The expression of the dimer-defective mutant induced enlarged mtDNA nucleoids, suggesting the importance of dimerization in the distribution of mtDNA. The reporter system also supported the association and mixture between independent nucleoids through TFAM by a cell fusion assay using hemagglutinating virus of Japan. We here, for the first time, visualized the interaction of TFAM molecules in mitochondria and proposed its implications for the dynamics of mtDNA nucleoids.

Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression.

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.

Multiple classes of transcription factors regulate the expression of VASCULAR-RELATED NAC-DOMAIN7, a master switch of xylem vessel differentiation.

The secondary cell walls of xylem cells, including vessel elements, provide mechanical strength and contribute to the conduction of water and minerals. VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a NAC-domain transcription factor that regulates the expression of genes required for xylem vessel element formation. Transient expression assays using 68 transcription factors that are expressed during xylem vessel differentiation showed that 14 transcription factors, including VND1-VND7, are putative positive regulators of VND7 expression. Electrophoretic mobility shift assays revealed that all seven VND proteins bound to the VND7 promoter region at its SMBE/TERE motif, indicating that VND7 is a direct target of all of the VND transcription factors. Overexpression of VND1-VND5, GATA12 and ANAC075, newly identified transcription factors that function upstream of VND7, resulted in ectopic xylem vessel element formation. These data suggest that VND7 transcription is a regulatory target of multiple classes of transcription factors.

Spin Fluctuations from Hertz to Terahertz on a Triangular Lattice.

The temporal magnetic correlations of the triangular-lattice antiferromagnet NiGa_{2}S_{4} are examined through 13 decades (10^{-13}-1 sec) using ultrahigh-resolution inelastic neutron scattering, muon spin relaxation, and ac and nonlinear susceptibility measurements. Unlike the short-ranged spatial correlations, the temperature dependence of the temporal correlations show distinct anomalies. The spin fluctuation rate decreases precipitously upon cooling towards T^{*}=8.5 K, but fluctuations on the microsecond time scale then persist in an anomalous dynamical regime for 4 K

Translational repression of the McKusick-Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites.

BACKGROUND: Upstream open reading frames (uORFs) are commonly found in the 5'-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick-Kaufman syndrome (MKKS) gene that causes a genetic disorder. METHODS: Northern blotting, 3'-RACE, and bioinformatics were used for determining the length of transcripts and their 3' ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization. RESULTS: The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5'-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5'-UTR are atypical but surely exist in human transcripts. CONCLUSIONS: Multiple uORFs at the 5'-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane. GENERAL SIGNIFICANCE: Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs.

Rapid selection of XO embryonic stem cells using Y chromosome-linked GFP transgenic mice.

We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9 % of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.

Mesoscopic structures of vermiculite and weathered biotite clays in suspension with and without cesium ions.

The effect of cesium (Cs) adsorption on the mesoscopic structure of the clay minerals vermiculite and weathered biotite (WB) in suspensions was elucidated by small-angle X-ray scattering (SAXS). The clay minerals form multilayered structures, and the Cs cations (Cs(+)) are strongly adsorbed in the interlayer space of the soil clays, in particular vermiculite and WB. SAXS was used to monitor the relationship between Cs(+) adsorption at the clay interlayers and the structural changes at length scales from 1 to 1000 Å. The variation in the distance between the neighboring clay sheets and the spatial arrangement of the clay sheets with and without Cs(+) were clarified. Our quantitative analyses revealed that the number of stacked layers of pure vermiculite was decreased by Cs(+) addition, whereas that of WB increased. Moreover, the average distance between the neighboring layers of vermiculite in suspension was larger than that of WB, which reflects the different conditions of Cs(+) intercalation. These findings provide fundamental insights that are important for predicting the environmental fate of radioactive Cs in contaminated regions and for developing methods for extracting Cs from soil.

Structure activity relationship study of curcumin analogues toward the amyloid-beta aggregation inhibitor.

Inhibition of the amyloid ß aggregation process could possibly prevent the onset of Alzheimer's disease. In this article, we report a structure-activity relationship study of curcumin analogues for anti amyloid ß aggregation activity. Compound 7, the ideal amyloid ß aggregation inhibitor in vitro among synthesized curcumin analogues, has not only potent anti amyloid ß aggregation effects, but also water solubility more than 160 times that of curcumin. In addition, new approaches to improve water solubility of curcumin-type compounds are proposed.

Synthesis and evaluation of curcumin derivatives toward an inhibitor of beta-site amyloid precursor protein cleaving enzyme 1.

To research a new non-peptidyl inhibitor of beta-site amyloid precursor protein cleaving enzyme 1, we focused on the curcumin framework, two phenolic groups combined with an sp2 carbon spacer for low-molecular and high lipophilicity. The structure-activity relationship study of curcumin derivatives is described. Our results indicate that phenolic hydroxy groups and an alkenyl spacer are important structural factors for the inhibition of beta-site amyloid precursor protein cleaving enzyme 1 and, furthermore, non-competitive inhibition of enzyme activity is anticipated from an inhibitory kinetics experiment and docking simulation.